Transcript Case 2: Schmidt lecture expanded

Using RNA sequencing to investigate
pulmonary endothelial glycocalyx
recovery after sepsis
Eric Schmidt, MD
Assistant Professor of Medicine
Pulmonary Sciences and Critical Care Medicine
Denver Health Medical Center
University of Colorado School of Medicine
The ancient riddle of sepsis
Majno G. JID 1991
Baron RM
AJRCMB 2005
Baron RM
AJRCMB 2005
Baron RM
AJRCMB 2005
How sepsis kills
Sepsis
Organ Failure
Mackenzie I , Lever A BMJ 2007;335:929-932
Severe Sepsis/
Septic Shock
Hospital mortality
Acute onset severe
hypoxemia
Noncardiogenic,
neutrophilic edema
High mortality,
morbidity
Commonly triggered
by sepsis
Acute respiratory
distress syndrome
(ARDS)
Have scientific advances translated
into clinical benefit?
• Many therapies have been developed to target
sepsis pathophysiology
– Anti-inflammatory therapies
• Corticosteroids
• Antibodies against inflammatory mediators
• Anti-pattern receptor agents
– Manipulation of coagulation/inflammation
All
failed!
• Antithrombin
• Activated protein C
• Sepsis remains most common cause of death in
ICUs worldwide
New ideas are needed!
• New targets
• New time points
The endothelial glycocalyx
Glycocalyx
EC
Goat coronary capillary
van der Berg et al. Circ Res 2003
Glycocalyx and the endothelial surface layer
• Glycocalyx
• ESL: the in vivo glycocalyx
VanTeeffelen et al. Trends Cardiovasc Med 2007
Measuring the endothelial glycocalyx
in mice
Ventilator
tubing
Heating
plate (titrated
to maintain
stable rectal
temperature)
Arterial
catheter for
MAP, CO
monitoring
IV line for drug,
colloid
administration
IV line for
continuous
anesthetic
infusion
ESL thickness (µm)
Sepsis and glycocalyx degradation
Sham
CLP
Saline
LPS
3
2.5
2
1.5
1
0.5
0
0
LPS = 20 mg/kg at t = 0
n = 3-6/group
0.5
1
1.5
24
Time (hours)
72
Relevance of ESL to lung inflammation
and injury?
VanTeeffelen et al. Trends Cardiovasc Med 2007
ESL degradation is necessary for septic
ARDS onset in mice
Schmidt et al. Nat Med 2012
ESL thickness (µm)
Is glycocalyx protection clinically
feasible?
Sham
CLP
Saline
LPS
3
2.5
2
1.5
1
0.5
0
0
LPS = 20 mg/kg at t = 0
n = 3-6/group
0.5
1
1.5
24
Time (hours)
72
Importance of glycocalyx recovery?
Nothing known!
ESL thickness (µm)
Sepsis and glycocalyx recovery?
Sham
CLP
Saline
LPS
3
2.5
2
1.5
1
0.5
0
0
LPS = 20 mg/kg at t = 0
n = 3-6/group
0.5
1
1.5
24
Time (hours)
72
Scientific question
• What transcriptional events occur within the
pulmonary microvasculature during glycocalyx
reconstitution?
Modeling septic lung injury in mice
• Cecal ligation and puncture
– “Gold standard” for polymicrobial sepsis (Rittirsch et al. Nat
Protocols 2009)
– Lung injury may be augmented by moderate (60%)
hyperoxia (Aggarwal et al. AJP-Lung 2010)
• “Double-hit” model with clinical relevance
Lung harvest and RNA extraction
• 48 hours after CLP/hyperoxia:
– Mice euthanized
– Pulmonary artery flushed with RNAlater
• Periphery of lung harvested (endothelium-rich)
• Kept in RNAlater overnight at 4 degrees C
• Controls: sham/hyperoxia x 48 hours
RNA quality control
RNA analysis screentape (Agilent): Provides RNA
quantification, quality data (“RIN”, 28s/18s)
RNA sequencing
• cDNA library made
– Coding mRNA isolated
– RNA fragmented
– Reverse transcriptase to cDNA
Shiroguchi K et al. PNAS 2012;109:1347-1352
©2012 by National Academy of Sciences
Shiroguchi K et al. PNAS 2012;109:1347-1352
©2012 by National Academy of Sciences
Output?
• Changes in entire
transcriptome
• Splice variants!
Analysis expectations
• Unbiased list of transcripts that are
differentially expressed in CLP and sham
– Hypothesis-generating
• Are certain biological pathways overrepresented?
– Heparan sulfate biosynthesis?
– Proteoglycans?